granzyme B CRISPR Activation Plasmid (h): sc-401469-ACT

Human GZMB encodes granzyme B, a trypsin-like serine protease stored in cytotoxic granules of CD8+ T cells and NK cells that mediates target-cell killing after perforin-dependent delivery. Granzyme B cleaves key substrates to activate caspase-dependent apoptosis and can also engage caspase-independent death pathways through mitochondrial perturbation, linking it to immune effector programs downstream of antigen recognition and inflammatory signaling. Dysregulated GZMB expression and activity have been associated with immune-driven tissue injury and remodeling in chronic inflammation and autoimmunity, as well as altered cytotoxic surveillance in cancer and viral infection contexts. As a result, GZMB is widely used as a functional marker for cytotoxic lymphocyte activation and as a mechanistic node for studying apoptosis, immune synapse biology, and immunopathology.

granzyme B CRISPR Activation Plasmid (h) provides a targeted, non-destructive approach to upregulating endogenous GZMB expression without altering the underlying DNA sequence.

granzyme B CRISPR Activation Plasmid (h) is a three-plasmid synergistic activation mediator (SAM) system engineered for highly efficient, site-specific transcriptional upregulation of the GZMB locus in human cell lines. The system is built around a catalytically inactive Cas9 (dCas9) carrying two inactivating mutations (D10A and N863A) that eliminate nuclease activity while preserving DNA binding. This dCas9 is fused to VP64, a potent transcriptional activator, and is co-expressed with a blasticidin resistance gene for selection. The second plasmid encodes the MS2-p65-HSF1 fusion protein, a secondary activator complex that works in concert with dCas9-VP64, alongside a hygromycin resistance gene. The third plasmid encodes a target-specific 20 nt sgRNA fused to two MS2 RNA aptamers that recruit the MS2-p65-HSF1 complex to the activation site, accompanied by a puromycin resistance gene. The three plasmids are delivered at a 1:1:1 mass ratio for balanced expression of all system components.

Once assembled at the target locus, the SAM complex binds within approximately 200 bp upstream of the GZMB transcriptional start site, where VP64, p65, and HSF1 act in concert to recruit transcriptional machinery and drive upregulation of endogenous granzyme B expression. Unlike nuclease-active Cas9, dCas9 does not introduce double-strand breaks or modify the genomic sequence, preserving the native GZMB locus and enabling the study of granzyme B-dependent transcriptional responses at the endogenous locus, making it a valuable tool for functional studies, target gene identification, and the modeling of granzyme B pathway restoration in tumor cells with silenced or reduced GZMB expression.

For Research Use Only. Not Intended for Diagnostic or Therapeutic Use.